The process of oxidation and the resulting damage to lipids, proteins and DNA have been implicated in the etiology of several diseases, but the strongest link is that between oxidized low density lipoprotein (LDL) and atherosclerosis. Factors which contribute to the oxidation of LDL include cigarette smoking, diet and diabetes. High density lipoprotein (HDL) is known to be protective against atherosclerosis, largely due to its role in the process of reverse cholesterol transport. It is reasonable to assume that the same physiologic and pathologic conditions that lead to oxidation of LDL will lead to oxidation of HDL. In fact, evidence that HDL protects LDL from oxidation suggests that HDL may be the more readily modified lipoprotein. This project is designed to test the hypothesis that oxidized HDL (oxHDL) exists in vivo and that oxidation of HDL results in the loss of its protective functional properties. The first aim is designed to document the presence of oxHDL in vivo in atherosclerotic lesions. The second aim is to reproducibly generate large quantities of oxHDL in vitro and to characterize modifications of the apoproteins with emphasis on structural changes in apoprotein A-I, the major protein component of HDL. The third aim is to examine the functional consequences of HDL oxidation in at least three functional systems. The capacity of oxHDL to mediate cellular cholesterol efflux and to activate the plasma cholesterol-esterifying enzyme, lecithin:cholesterol acyltransferase, will be tested. These two functions are critical components of reverse cholesterol transport. The effect of oxidation on HDL delivery of cholesterol to ovarian steroidogenic cells will be examined. Documentation of decreased HDL function resulting from oxidative damage will add a new dimension to our understanding of the role of lipoprotein oxidation in atherogenesis and cholesterol metabolism. The final aim is to document the presence of oxHDL in human plasma using a monoclonal antibody to oxidized human HDL or the presence of auto antibodies to oxHDL. The unifying hypothesis that oxidation of lipoproteins plays a pivotal role in the progression of atherosclerosis has focused entirely on oxLDL. Data implicating oxHDL as well in the disease etiology will strengthen this unifying hypothesis.